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At least 19 recordsLinked to original sources
PubMedDate not supplied

In vitro metabolism of GV150013X by using liver microsomes and liver tissue slices from different species.

The metabolism of GV150013X was studied in vitro using washed liver microsomes and liver tissue slices from different species. This work was carried out in order to compare the metabolite profiles resulting from incubation of GV150013X with human, rat, dog and rabbit liver microsomes and those from rat, rabbit and human liver tissue slices. This compound was found to be converted to at least 8 metabolites by rat and human liver microsomes. In rabbit liver microsomes, the three metabolites M4, M7 and M8, and in dog liver microsomes metabolite M1, were not detected. The main metabolites, M2, M5 and M6, were present in human, rat and rabbit liver tissue slices, while the three metabolites M3, M4 and M8 and metabolite M1 were not detected in rabbit and rat liver tissue slices, respectively. In rat liver tissue slices, the major metabolites plus five minor metabolites, one sulphate conjugate of a monohydroxylated, three trihydroxylated and one dihydroxylated were identified based on HPLC retention time and thermospray-mass spectrometry data. Quantitative species differences among rat, dog, rabbit and human were observed, while qualitative differences only between rabbit and other species were detected.

Adamantane↗
PubMed1996

A convenient method to discriminate between cytochrome P450 enzymes and flavin-containing monooxygenases in human liver microsomes.

Liver microsomes are a frequently used probe to investigate the phase I metabolism of xenobiotics in vitro. Structures containing nucleophilic hetero-atoms are possible substrates for cytochrome P450 enzymes (P450) and flavin-containing monooxygenases (FMO). Both enzymes are located in the endoplasmatic reticulum of hepatocytes and both need oxygen and NADPH as cofactors. The common method to distinguish between the two enzyme systems is to use the thermal inactivation of FMO and to inhibit P450 completely with carbon monoxide, N-octylamine or N-benzylimidazole. In the literature no indication could be found that the heat inactivation of FMO does not affect any of the human P450 enzymes or that the overall P450 inhibitors inhibit the different human P450 enzymes sufficiently and do not affect the FMO. The effect of N-benzylimidazole and heat inactivation was tested on specific activities of seven P450 enzymes in human liver microsomes, 1A2, 2A6, 2C9, 2C19, 2D6, 3A4/5, and 2E1, using methoxyresorufin O-demethylation, coumarin 7-hydroxylation, (S)-warfarin 4-hydroxylation, (S)-(+)-mephenytoin 4-hydroxylation, dextrometorphan O-demethylation, oxidation of denitronifedipine, and chlorzoxazone 6-hydroxylation respectively. The sulfoxidation of methimazole (MMI) was used as a specific probe for the determination of FMO activity. Methimazole sulfoxidation was compared with the well known assay for FMO metabolism, the formation of N,N-dimethylaniline (DMA) N-oxide, to be confirmed as an exclusively FMO mediated reaction. The participation of P450 and FMO in the sulfoxidation of four sulfur containing peptides, ametryne; terbutryne, prometryne and methiocarb was investigated using human liver microsomes. All four reactions were demonstrated to be catalysed predominantly by cytochrome P450.

Aniline Compounds↗
PubMed1992

Degradation of methyl and ethyl mercury into inorganic mercury by hydroxyl radical produced from rat liver microsomes.

Liver microsomes were prepared from Wistar rat by the Ca2+ aggregation method. Under various conditions, ethyl mercury chloride (EtHgCl) or methyl mercury chloride (MeHgCl) was incubated with the microsomal preparations. After the incubation, the amounts of inorganic Hg and hydroxyl radical (.OH) in the preparations were determined. Although the preparations alone produced a small amount of inorganic Hg and .OH, the addition of NADPH to the preparations increased both inorganic Hg and .OH production, which were further accelerated by the addition of KCN. The addition of Fe(III)EDTA, a .OH formation promoter, to the microsome-NADPH-KCN system increased inorganic Hg production, whereas the addition of diethylenetriamine pentaacetic acid, a .OH formation inhibitor, decreased inorganic Hg production. When .OH scavengers such as mannitol and dimethyl sulfoxide were added to this system, the inorganic Hg production decreased. These results suggested that the .OH produced from liver microsomes was responsible for the degradation of MeHg and EtHg. Since both .OH and inorganic Hg production decreased with a concomitant decrease in NADPH-cytochrome P-450 reductase activities, it is suggested that this enzyme may be involved in the microsomal degradation of MeHg and EtHg.

Animals↗
PubMed2001

A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4'-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6beta-hydroxylation.

Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19); dextromethorphan O-demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6beta-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6beta-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.

Animals↗
PubMed1996

Hydroxylation of carbon atoms of the alkyl chain of symmetrical N-nitrosodialkylamines by rat liver microsomes.

Liver microsomal preparations from control and treated rats (cytochromes P450 1A, 2B, 3A and 2E1-induced) metabolized at variable metabolic rates three nitrosodialkylamines (N-nitroso-dipropyl, dibutyl and diamyl-amines) into aldehydes and hydroxy-nitrosamines. The longer the alkyl chain, the smaller was the metabolic rate of the alpha-hydroxylation of alkyl chain yielding aldehyde and the greater was the metabolic rate of the corresponding (omega-1)-hydroxyl metabolite formation. Thus, the (omega-1) hydroxylation of the alkyl chain was the major metabolic pathway of N-nitrosodiamylamine (NDAA) so far as it represented 22-fold the alpha-hydroxylation. The balance between beta to omega hydroxylation and alpha-hydroxylation depends upon the alkyl chain length and also on specific P450 isoform induction.

Animals↗
PubMed1979

Synthesis and identification of products derived from the metabolism of the carcinostatic 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea by rat liver microsomes.

Liver microsomal metabolism of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea in the presence of reduced nicotinamide adenine dinucleotide phosphate and O2 was shown to produce seven metabolites that included the parent urea. A cytochrome P-450-dependent monohydroxylation of the cyclohexyl ring occurred in 3 positions, cis-3, trans-3, and cis-4, and on the methyl group to form a trans-4-hydroxymethyl derivative. In addition, monohydroxylation of the 2-chloroethyl carbon attached to the N-1 urea nitrogen yielded an alpha-hydroxy metabolite. A ring-hydroxylated derivative remained unidentified while the structures of all other such derivatives were established by comparison with compound synthesized, purified by high-pressure liquid chromatography, and characterized by mass spectral and nuclear magnetic resonance analyses. It was tentatively concluded that some parent urea is formed by a cytochrome P-450 dependent reaction because of a requirement for reduced nicotinamide adenine dinucleotide phosphate and inhibition by CO. Microsomes from rats pretreated with phenobarbital showed about a 3-fold increase in hydroxylation rate while phenobarbital-treated mice microsomes were induced 8-fold. However, in both species, the induced hydroxylation rate was about 4 nmol/min/mg protein. When microsomes from phenobarbital-induced rats were used, a mixture of 80% CO:20% O2 decreased the rate of formation of all metabolites to 14% of that in 80% N2:20% O2.

Animals↗
PubMedDate not supplied

Rates of phencyclidine metabolism by the isolated perfused lung, lung microsomes, and liver microsomes of rabbits.

The metabolism of phencyclidine (PCP) was examined in the isolated perfused lung (lPL), lung microsomes, and liver microsomes of rabbits. The concentration of PCP in the IPL perfusate decreased rapidly with concomitant increases in the concentrations of PCP metabolites. The total rate of PCP metabolite appearance in the perfusate of the IPL was 77.4 nmol/lung/min. The apparent Vmax of PCP metabolism by the lung and liver microsomes were 1.52 and 22.9 nmol of PCP metabolised per mg of microsomal protein per min, respectively. Extrapolated to the whole lung, the apparent Vmax of PCP metabolism by lung microsomes was equivalent to 78.6 nmol of PCP metabolized per lung per min, essentially the same as that for the IPL.

Animals↗
PubMed1984

Induction of laurate omega-hydroxylase by di (2-ethylhexyl)phthalate in rat liver microsomes.

Liver microsomes, prepared from rats fed a diet containing di (2-ethylhexyl)phthalate, were observed to hydroxylate lauric acid at carbon 12 at a specific activity 6 times greater than control rats. There was no increase in the specific activity of the laurate 11-hydroxylase. The specific activity of hepatic microsomal NADPH-cytochrome c reductase was increased 2-fold by phthalate feeding, but no effect was observed in the specific contents of cytochromes b5 or P-450. These results indicate that di (2-ethylhexyl)phthalate is similar to the hypolipidemic agent and peroxisomal proliferator , clofibrate, which was previously reported to be a novel inducer of the cytochrome P-450 mediated omega-hydroxylation of fatty acids.

Animals↗
PubMed1983

Solubilization and partial purification of two forms of cytochrome P-450 from trout liver microsomes.

Liver microsomal cytochrome P-450 content, NADPH-cytochrome c reductase, aniline 4-hydroxylase and ethylmorphine N-demethylase activities of rainbow trout, Salmo gairdneri, were found to be 0.16 (n = 10) nmol P-450/mg protein, 38 (n = 5) units/mg protein, 0.04 (n = 4) nmol p-aminophenol/min/mg protein and 0.174 (n = 3) nmol formaldehyde/min/mg protein, respectively. Trout liver cytochrome P-450 was solubilized by treatment of microsomes with sodium cholate. Chromatography on DEAE-cellulose column yielded two distinct cytochrome P-450 fractions from solubilized microsomes. Cytochrome P-450-I was eluted with Emulgen 913-containing buffer. Application of 0.08 M KCl in Emulgen 913-containing buffer to the DEAE-cellulose column eluted cytochrome P-450-II fraction. Cytochrome P-450-I was further purified on hydroxylapatite column. CO-difference spectrum of dithionite-reduced cytochrome P-450-I gave a peak at 449 nm while the similar spectrum of cytochrome P-450-II showed a maximum absorbance at 451 nm. Monomer molecular weights of cytochrome P-450-I and cytochrome P-450-II were determined by sodium dodecyl sulfate gel electrophoresis and found to be 56,000 and 48,500, respectively.

Animals↗
PubMedDate not supplied

Influence of pregnancy and folic acid on phenytoin metabolism by rat liver microsomes.

Liver microsomal suspensions from pregnant and nonpregnant Sprague-Dawley rats were assayed for NADPH-dependent metabolism of phenytoin to the hydroxylated products, 5-(rho-hydroxyphenyl)-5-phenylhydantoin (rhoHPPH) and 5-(3,4-dihydroxy-1,5-cyclohexadien-1-yl)5-phenylhydantoin (H2DIOL). UDP-glucuronic acid-dependent glucuronyltransferase activity for conjugating rhoHPPH was also measured. The specific activity for formation of rhoHPPH decreased by 35% in 21-day pregnant rats compared with nonpregnant rats, whereas specific activity for production of H2DIOL was increased approximately 2-fold on the 7th and 14th but not the 21st day of pregnancy. Because of a 25% increase in liver weight during pregnancy, the total hepatic phenytoin hydroxylase activity in pregnant rats (day 21) was not significantly different from that of nonpregnant controls. Folic acid treatment during pregnancy prevented the decrease in phenytoin hydroxylase specific activity. The KM for production of either rhoHPPH or H2DIOL was similar (90 micrometer) in pregnant (day 21) and nonpregnant animals. Thus, overall hepatic microsomal enzyme activity for metabolizing phenytoin was not significantly reduced during pregnancy in rats, although the ratio of H2DIOL to rhoHPPH was increased during the first two weeks. Folic acid may play a role in pregnancy-associated changes in phenytoin hydroxylase activity.

Animals↗
PubMedDate not supplied

Effects of pesticides on metabolism of steroid hormone by rodent liver microsomes.

Liver microsomal steroid hydroxylating enzymes and prostatic testosterone-5alpha-reductase were studied in rat and mouse. Organochlorine and organophosphate pesticides tended to inhibit liver steroid hydroxylations, while carbofuran slightly stimulated them. Neither species was consistently more sensitive to pesticide effects than the other. All the pesticides bound to cytochrome P-450, producing Type I spectral changes. Values of Ks ranged from 1.9 to 8.7 mM for organochlorine and organophosphate compounds. Affinity for carbofuran was much lower (Ks=100-200mM).

Animals↗
PubMed1997

In vitro glucuronidation of D-23129, a new anticonvulsant, by human liver microsomes and liver slices.

1. The metabolic profile of D-23129, a new anticonvulsant agent, was studied in vitro using human liver microsomes and fresh liver slices. 2. Oxidative metabolism appeared to be minimal with D-23129. The percent mean total radioactivity not associated with the parent compound recovered from oxidative metabolism studies from three individual liver donors was 0.7% +/- 0.6 SD and was not significantly different from [14C]-D-23129 incubated with heat inactivated microsomes, mean = 0.5% +/- 0.4 SD. 3. Phase II conjugation dominated the metabolism of D-23129 producing two distinct N-glucuronides as the primary metabolites. These metabolites were identified by electrospray ionization LC/MS. 4. The apparent Km for one of the glucuronide metabolites was determined in human liver microsome preparations from two individual liver donors to be 131 and 264 microM respectively, Vmax determined for the same microsomal preparations yielded 48.9 and 59.9 pmol/min/mg protein.

Anticonvulsants↗
PubMed1975

Effect of rifampicin treatment on the metabolism of oestradiol and 17alpha-ethinyloestradiol by human liver microsomes.

Liver biopsies were obtained from four patients treated with rifampicin 600 mg for 6-10 days. Hepatic microsomes were incubated with an NADPH-regenerating system and the substrates [2,4,6,7-3H] oestradiol, [6,7-3H] oestradiol, [2,4,6,7-3H] ethinyloestradiol and [6,7-3H] ethinyloestradiol. The hydroxylation rates of these steroids at the labelled positions of rings A and B were determined by measuring the transformation of tritium into HTO by the microsomal enzymes. Comparison with previously published data showed that treatment with rifampicin caused a fourfold increase in the rate of hydroxylation of oestradiol and ethinyloestradiol at positions C-2/C-4 of ring A and C-6/C-7 of ring B. The acceleration of oestrogen hydroxylation by rifampicin was paralleled by an increase in microsomal cytochrome P-450, and also by microsomal reduction of rifampicin-quinone, a reactive metabolite of rifampicin. The increased aromatic hydroxylation of oestradiol and ethinyloestradiol leads to enhancement of their irreversible binding to microsomal protein. The data provide an explanation for the diminished efficacy of oestrogens in contraceptive formulations given to patients under treatment with rifampicin.

Adult↗
PubMedDate not supplied

Metabolism and covalent binding of [14C]toluene by human and rat liver microsomal fractions and liver slices.

The in vitro metabolism of [14C]toluene by liver microsomes and liver slices from male Fischer F344 rats and human subjects has been compared. Rat liver microsomes produced only benzyl alcohol from toluene. Liver microsomes from human subjects metabolized toluene to benzyl alcohol, benzaldehyde, and benzoic acid. Liver microsomes from one human donor also produced p-cresol and o-cresol. The overall rate of toluene metabolism by human liver microsomes was 9-fold greater than by rat liver microsomes. Human liver microsomal metabolism of benzyl alcohol to benzaldehyde required NADPH and was inhibited by carbon monoxide and high pH (pH 10). but was not inhibited by ADP-ribose or sodium azide. These results suggest that cytochrome P-450, rather than alcohol dehydrogenase, was responsible for the metabolism of benzyl alcohol to benzaldehyde. Human and rat liver slices metabolized toluene to hippuric acid and benzoic acid. The overall rate of toluene metabolism by human liver slices was 1.3-fold greater than by rat liver slices. Cresols and cresol conjugates were not detected in human or rat liver slice incubations. Covalent binding of [14C]toluene to human liver microsomes and slices was 21-fold and 4-fold greater than to the comparable rat liver preparations. Covalent binding did not occur in the absence of NADPH, was significantly decreased by coincubation with cysteine, glutathione, or superoxide dismutase, and was unaffected by coincubation with lysine. Protease and ribonuclease digestion decreased the amount of toluene covalently bound to human liver microsomes by 78% and 27% respectively. Acid washing of human liver microsomes had no effect on covalent binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Drinking↗
PubMed1993

An endogenous glycogen-associated compound modulates glucose-6-phosphatase activity in rat liver microsomes.

Liver glycogen is closely associated with the endoplasmic reticulum, which contains the glucose-6-phosphatase enzyme system that catalyses the final step of hepatic glucose production. To examine whether this structural association has functional consequences, microsomes were isolated from 48 h fasted (n = 6) and ad lib fed rats (n = 3). Microsomes from fed rats had a higher glycogen content and lower enzyme activity than fasted rats. Overall, glucose-6-phosphatase activity was inversely proportional to microsomal glycogen content. Partially purified rabbit or rat liver glycogen, at physiological relevant concentrations (10-100 mM glucose equivalents), added directly to either intact or Triton-disrupted microsomes from fasted rats significantly decreased glucose-6-phosphatase activity. Inhibitory activity was present in native liver glycogen prepared by sedimentation and could be dissociated from glycogen by ion-exchange and ultrafiltration. These findings suggest that a low molecular weight (< 5000 D) compound closely associated with glycogen can modulate glucose-6-phosphatase and may have a physiologic role in the regulation of hepatic glucose production.

Animals↗
PubMed2006

Reduction of Nomega-hydroxy-L-arginine to L-arginine by pig liver microsomes, mitochondria, and human liver microsomes.

Nomega-Hydroxy-L-arginine, the intermediate in nitric oxide formation from L-arginine catalyzed by NO synthase, can be released into the extracellular space. It has been suggested that it can circulate and exert paracrine effects. Since it cannot only be used as substrate by NO synthases, but can also be oxidized by cytochrome P450 and other hemoproteins in a superoxide-dependent manner, it has been proposed that it can serve as NO donor. In the present study, the in vitro reduction of Nomega-hydroxy-L-arginine was examined. Pig and human liver microsomes as well as pig liver mitochondria were capable of reducing Nomega-hydroxy-L-arginine to L-arginine in an oxygen-insensitive enzymatic reaction. These results demonstrate that this metabolic pathway has to be considered when suggesting Nomega-hydroxy-L-arginine as NO-precursor. The reconstituted liver microsomal system of a pig liver CYP2D enzyme, the benzamidoxime reductase, was unable to replace microsomes to produce L-arginine from Nomega-hydroxy-L-arginine.

Animals↗